PROJECT SUMMARY/ABSTRACT Development of an immunoengineered technology to selectively stimulate and expand regulatory T (TReg) cells in vivo would be transformative for autoimmune disease treatment and transplantation medicine. Extensive evidence has established that adoptively transferred TReg cells, and in particular antigen-specific TReg cells, effectively suppress pathogenic autoimmune activity to combat disease. However, logistical and manufacturing complications as well as safety concerns associated with ex vivo expanded T cells impede clinical adoption of this approach. Thus, there is an unmet medical need for an off-the-shelf, non-cellular platform that activates antigen-specific TReg cells to manifest immunosuppression directly in patients. The interleukin-2 (IL-2) cytokine potently activates TReg cells, and has proven to be a promising alternative to adoptive TReg cell transfer. However, IL-2 concurrently stimulates activation of effector cells, resulting in harmful off-target effects and toxicities. Co- administration of IL-2 in complex with certain anti-cytokine antibodies preferentially activates and expands TReg over effector cells, but concerns about cytokine/antibody complex dissociation hinder the therapeutic development of this approach. Furthermore, IL-2 treatment induces non-specific expansion of TReg cells, with limited enrichment of therapeutically superior antigen-specific cells. This proposal aims to develop a robust and versatile platform that activates antigen-specific TReg cells and simultaneously inhibits the function of pathogenic autoreactive T cells. This technology synthesizes, for the first time, three design approaches: (1) Selective delivery of IL-2 to TReg cells; (2) Stimulation of antigen-specific TReg cells using artificial antigen-presenting cells (aAPCs); and (3) Localized release of an immunosuppressive drug to prevent effector T cell activation. The novel platform, denoted TolAPC, comprises nanoparticles coated with self peptide-loaded major histocompatibility complex (MHC) as well as a stabilized single-chain fusion format of an IL-2/antibody complex that selectively promotes TReg expansion. These particles are also engineered for controlled release of the immunosuppressive drug rapamycin. TolAPCs will be characterized in vitro and in vivo to assess selective expansion of TReg cells that can suppress pathogenic self-reactive effector T cells. They will also be therapeutically evaluated in autoimmune disease studies in mice. As a model system, TolAPC activity will be assessed in type 1 diabetes (T1D), a chronic autoimmune disease that is a growing threat to global health with incidence rising at an alarming rate of 3% annually. The modularity of the TolAPC platform will allow for ready extension to a host of applications in autoimmune disease treatment and transplant tolerance through substitution of the antigen specificity. Overall, our targeted immunomodulatory nanotechnology will serve as a powerful and general tool for biasing immune activation to effect therapeutically relevant outcomes.